ApoE receptors mediate many of the functions of apoE in the CNS; including endocytosis of lipoproteins, activation of kinase signaling pathways, and modulation of synaptic signaling and glial activation. One important aspect of apoE receptor biology is its shedding from the cell via regulated cleavage mechanisms. In this project, we will focus on one of these apoE receptors, ApoER2. In Aim 1, we will test how proteolytic enzymes generate soluble receptors and cytoplasmic fragments, analyzing ADAMs and BACE. We will determine the mechanism of these cleavages, testing whether they are altered by the alternatively spliced O- linked glycosylation domain of ApoER2 and inhibited by the endogenous metalloproteinase inhibitor, TIMP-3. We hypothesize that the proteases responsible for APR cleavage also act on apoE receptors. In Aim 2, we will focus on the regulation of these cleavage events. We will test how cytoplasmic adaptor proteins (FE65, Dab-1, SNX-17), influence trafficking of ApoER2, and how ligand binding (specifically apoE isoforms) affect receptor metabolism. We will examine the role shedding plays in the functions of ApoER2 in endocytosis and signaling, inlcuding mechanisms of neurotoxicity. In Aim 3, we will test how secreted apoE receptors are cleared, examining several possible mediators of endocytosis. Through these three aims, we will define the production, function, and clearance of soluble ApoER2, and how the shedding process affects the functions of full-length ApoER2. In Aim 4, we will examine the connections between ApoER2 and APP, using primary neurons and genetically modified mice. We will test how ligands (apoE-lipoproteins) affect ApoER2 proteolysis in vivo, and how soluble and full length ApoER2 affect AH plaque formation. We hypothesizethat because APP and apoE receptors share ligands, adaptor proteins, and proteolytic pathways, alteration of receptors such as ApoER2 have important effects on APP trafficking and proteolysis in vivo. We will work in concert with the other projects to progress each of these aims: Project 1for understanding how physiologically relevant forms of human apoE isoforms affect ApoER2 function including neurotoxicity; Project 2 for analysis of receptor splice variants; Project 3 for analysis of ApoER2 trafficking and processing; and Project 5 for determining the effects of ApoER2 on neuronal signaling and APP processing in vivo. Overall, these studies will examine the functional effects of apoE isoforms via ApoER2 in the CNS.